Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from Increased myoepithelial cells of bronchial submucosal in fatal asthma F H Y Green,1 D J Williams,1 A James,2,3 L J McPhee,1 I Mitchell,1 T Mauad4 c Additional data are published ABSTRACT secreted into the mucous ducts and from online only at http://thorax.bmj. Background: Fatal asthma is characterised by enlarge- there expelled into the airway lumen. com/content/vol65/issue1 ment of bronchial mucous glands and tenacious plugs of Myoepithelial cells are ubiquitous components 1 Respiratory Research Group, in the airway lumen. Myoepithelial cells, located of exocrine glands. They lie between the basement Faculty of Medicine, University within the mucous glands, contain contractile proteins membrane and the basal surface of the acinar cells of Calgary, Alberta, Canada; 2 Department of Pulmonary which provide structural support to mucous cells and and have ‘‘octopus-like’’ branching processes which 10 Physiology, West Australian actively facilitate glandular secretion. extend between the secretory epithelial cells. Sleep Disorders Research Objectives: To determine if myoepithelial cells are They have been studied in the salivary, mammary, Institute, Queen Elizabeth II increased in the bronchial submucosal glands of patients prostate, lachrymal and sweat glands in several Medical Centre, Perth, Western species.10–12 Meyrick and Reid13 described the Australia, Australia; 3 School of with fatal asthma. Medicine and Pharmacology, Methods: Autopsied lungs from 12 patients with fatal anatomical features of myoepithelial cells beneath University of Western Australia, asthma (FA), 12 patients with asthma dying of non- the serous, mucous and collecting cells of Perth, Western Australia, human bronchial submucosal glands. However, the 4 respiratory causes (NFA) and 12 non-asthma control Australia; Laboratory of Air mechanism of mucus expulsion from the glandular Pollution, Department of cases (NAC) were obtained through the Prairie Provinces Pathology, Sao Paulo University Asthma Study. Transverse sections of segmental bronchi acini has not been studied in human bronchial Medical School, Sao Paulo, SP, from three lobes were stained for mucus and smooth mucous glands. Brazil muscle actin and the area fractions of mucous plugs, Myoepithelial cells are contractile in nature, mucous glands and myoepithelial cells determined by possessing myofilaments composed of actin and Correspondence to: 10 13 14 point counting. The fine structure of the myoepithelial myosin. Their contraction contributes to Dr F H Y Green, Pathology and 14 Laboratory Medicine, Faculty of cells was examined by electron microscopy. glandular secretion. The mechanism of stimula- Medicine, University of Calgary, Results: FA was characterised by significant increases in tion varies by gland type and species. Contraction 3330 Hospital Drive NW, mucous gland (p = 0.003), mucous plug (p = 0.004) and may be caused by cholinergic, adrenergic or non- Calgary, Alberta T2N 4N1, 15 Canada; [email protected] areas (p = 0.017) compared with NAC. adrenergic/non-cholinergic mechanisms. When the ratio of myoepithelial cell area to total gland As the area of the submucosal glands is increased 3 5–7 Received 11 December 2008 area was examined, there was a disproportionate and in fatal asthma, it is reasonable to assume that Accepted 18 October 2009 the myoepithelial cell network is also increased in significant increase in FA compared with NAC http://thorax.bmj.com/ Published Online First relation to the increase in gland size. Given the 8 December 2009 (p = 0.014). Electron microscopy of FA cases revealed hypertrophy of the myoepithelial cells with increased striking amount of mucus found at autopsy in the airways in status asthmaticus and the marked intracellular myofilaments. The NFA group showed 47 changes in these features that were intermediate increase in airway smooth muscle, we hypothe- between the FA and NAC groups but the differences were sised that contractile elements are disproportio- not significant. nately increased within the mucous glands in fatal Conclusions: Bronchial mucous glands and mucous asthma. We report that this is the case—a finding

that has important implications for understanding on September 25, 2021 by guest. Protected copyright. gland myoepithelial cell smooth muscle actin are and preventing death from asthma. increased in fatal asthma and may contribute to asphyxia due to mucous plugging. METHODS Subjects and study design The World Health Organization estimates that The study was based on autopsy materials 255 000 people died of asthma in 2005 and that 300 collected for the Prairie Provinces Asthma Study million people are currently affected by asthma.1 (PPAS), a multicentre study of asthma fatalities Death occurs by asphyxiation owing to airway occurring in Alberta, Saskatchewan and Manitoba closure through bronchoconstriction and mucous from November 1992 to October 1995.16 Cases plugging on a background of inflammation and were defined as subjects dying of asthma (fatal airway wall remodelling.2–4 Research has focused asthma, FA). There were two control groups: a more on the changes in airway smooth muscle and non-fatal asthma (NFA) group comprising subjects less on the enlarged mucous glands and tenacious with a history of asthma but who had died of non- mucous plugs.3 5–7 Overproduction of mucus in respiratory causes and a non-asthma control patients with asthma stems from a combination of (NAC) group with no history of asthma or other hyperplasia of goblet cells and enlargement of the respiratory disease at death. Deaths occurring in bronchial submucosal glands.5 The mucus in individuals with a history of asthma were reported asthma has altered viscoelastic and biochemical to the study team through the medical examiners properties that contribute to adhesivity and or coroner’s offices, hospitals and provincial vital impaired clearance.89In asthma there is an increase statistics departments. Provincial departments of in the ratio of mucous to serous cells within the vital statistics were contacted every 3 months to mucous glands.5 Mucus produced in the glands is ensure that no deaths classified as asthma in this

32 Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from age range were missed. NAC cases were obtained from the lumen, interstitium and blood vessels. These area fractions were Alberta Medical Examiner’s Office and local hospitals. The estimated as the number of points falling on the feature of criteria used by the pathologists and clinicians to classify the interest divided by the total number of points falling on the cases and control subjects, as well as the inclusion/exclusion mucous gland. criteria, are available in the online supplement. Further details of the morphometric methods are given in the After notification of death, the study team contacted the next online supplement. of kin to obtain consent for autopsy. The next of kin were asked to complete a questionnaire that sought information on asthma Electron microscopy severity, age of onset, duration of asthma, asthma medications Tissue for ultrastructural analysis was obtained from four and smoking history. Those with a history of asthma were subjects who had died of asthma (FA) and underwent autopsy assigned a category for asthma severity, unrelated to the cause at the Department of Pathology, Sa˜oPaulo University between of death, based on 2006 guidelines from the Global Initiative for 2005 and 2007. All had a history of asthma and no other lung 17 Asthma. Subjects were classified as having severe asthma if disease. Clinical data including treatment, smoking habits, they were using oral corticosteroids, reported hospitalisations duration of disease and previous hospitalisations were obtained for asthma (ever) or had daily symptoms. Subjects who had by administering a questionnaire to the relatives.19 Three none of the above but had symptoms on most days or nights control subjects were studied, all non-smokers with no history (more than 3 days per week), used regular inhaled corticoster- of asthma, wheeze, use of asthma medications or other lung oids or used reliever medications on most days or nights were disease and no gross or microscopic evidence of asthma at classified as having moderate asthma. All other cases were autopsy. classified as mild. Small (26262 mm) fragments of lobar bronchial wall were fixed in 2% glutaraldehyde dissolved in 0.15 M phosphate buffer Tissue sampling for light microscopy and morphometry at pH 7.2 for 1 h, followed by post-fixation in 1% osmium Left lungs taken at autopsy were fixed in inflation via the blood tetroxide dissolved in 0.9% sodium chloride for 1 h and vessels and airways with glutaraldehyde fixative (2.5% in 0.1 M embedded in Araldite resin. Ultrathin sections were studied phosphate buffer, pH 7.3). The airway pressure was with a transmission electron microscope. 20 cm H2O. The dual fixation method was developed to circumvent the poor airway perfusion resulting from mucous Statistical analysis plugs in the FA group. Transverse sections from segmental A total of 108 cases and controls had been accessioned to the bronchi were used from three sites: left upper lobe (LUL), left PPAS. A subset of 36 cases (12 FA, 12 NFA, 12 NAC) was used in lower lobe anterior bronchus (LAB) and left lower lobe posterior this study. This sample size was based on preliminary data for bronchus (LPB). Segmental bronchi were selected because they airway smooth muscle which revealed that 12 cases per group contained the greatest proportion of mucous glands relative to would provide sufficient power (77%) to detect a significant airway wall size. Tissue blocks were embedded in paraffin wax

effect. Further information regarding the power calculation is http://thorax.bmj.com/ and sections stained with haematoxylin and eosin (H&E) and available in the online supplement. From the 108 subjects we Alcian blue/PAS (AB/PAS) at pH 2.5 for characterisation of randomly selected 12 subjects per group such that each group mucous plugs and mucous cells. Sections from the LPB were had equal numbers of men and women, and smokers and non- immunostained for smooth muscle actin (DAKO Immunostain) smokers. For example, the FA group had six men (3 smokers and for identification of myoepithelial cells. More detail of the 3 non-smokers) and six women (3 smokers and 3 non-smokers). fixation techniques and sampling sites are available in the online This selection was done to provide balanced groups to test the supplement. relationship between the primary variable of interest, myo-

epithelial cell size, and asthma group (severity). A comparison of on September 25, 2021 by guest. Protected copyright. Morphometry the subgroup of 36 cases in this study with the total population (n = 108) showed no significant differences between the two The area fractions of components of the airway wall and groups for the demographic variables of age, time from death to mucous glands were determined by point counting using a autopsy, age at onset of asthma, asthma duration and (for Zeiss-Axioplan light microscope, drawing tube and square smokers) pack years. lattice grid containing 240 points.18 For group comparisons (FA, NFA, and NAC), normality Structures identified and quantified by area on the H&E assumptions were tested and the appropriate parametric and stained sections of the whole bronchus included bronchial non-parametric tests were used accordingly. For comparison mucous glands and the bronchial airway lumen (subdivided into between groups, one-way ANOVA with post hoc Tukey- free lumen and mucus/cells). The perimeter of the airway (Pbm) Kramer multiple comparison or Student t tests were used for was determined on the same grid by counting the number of continuous variables. Pearson x2 and Fisher exact tests were intersections between the grid lines and the luminal aspect of used for categorical data. A p value of 0.05 or less was considered the epithelial basement membrane (laminar reticularis).4 To significant. normalise the mucous gland area to airway size, the mucous gland area was divided by the Pbm. The area of the airway lumen occupied by mucous plugs was calculated by dividing the RESULTS mucous plug area by the measured total airway luminal area Patient characterisation and expressed as a percentage. Mucous gland duct ectasia was The demographic characteristics and the causes of death of the graded on the H&E stained sections on a 5-point scale (0–5) of PPAS study groups are shown in table 1. No significant increasing severity. differences were observed between the FA and NFA groups for Subcomponents of the mucous glands were measured on the age at death, duration of asthma or age at onset of asthma. sections stained for alpha smooth muscle actin. These included Asthma severity was significantly greater in the FA group than myoepithelial cells, acinar cells (nuclei and cytoplasm), acinar in the NFA group (p = 0.032, Pearson x2 test). This was reflected

Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 33 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from in the increased use of oral corticosteroids or short-acting Electron microscopy bronchodilators in the FA group compared with the NFA group In the FA and NAC cases, myoepithelial cells were positioned (table 1). Inhaled corticosteroid use was similar for the FA and between the epithelial cells and the basement membrane of the NFA groups. For those subjects who smoked, the NFA group submucosal glands; the latter was thickened in the acini of the had significantly (p = 0.04) greater pack years than the FA subjects with asthma. The myoepithelial cell cytoplasm group. The mean time between death and autopsy for the three contained dense bodies and myofilaments which lay parallel groups was not significantly different (table 1). to the long axis of the cell. The nuclei were oval or elongated. The demographic information for the subjects used for The myoepithelial cells were attached to the acinar gland cells electron microscopy from Sao Paulo, Brazil is summarised in with desmosomes. In FA the myoepithelial cells had more table 2. myofilaments and more prominent cytoplasmic processes extending between the mucous cells (figs 2 and 3). These changes were not quantified. The subjects with asthma had increased airway smooth muscle, Morphometry mucous gland enlargement, mucous duct ectasia, Airway size (Pbm) did not differ significantly between the three hyperplasia of the lining , thickening of the groups (table 3). Mucous gland area was increased (p = 0.003) in subepithelial collagen layer and infiltration of eosinophils and the FA group compared with the NAC group when data from all lymphocytes in the bronchial wall. In addition, most of the FA three lobes were included in the analysis. cases had extensive mucous plugs and bronchoconstriction. The average normalised values of subcomponents of the NAC cases had no features of asthma. mucous glands were increased in the FA group compared with Sections stained for smooth muscle actin showed staining of NFA and NAC (table 4). However, only myoepithelial actin in bronchial smooth muscle, vessels and myoepithelial cells of the the FA group was significantly increased (p = 0.017) compared bronchial mucous glands (fig 1). The latter were located with the NAC group. When the ratio of myoepithelial cell area between the basal surface of the glandular epithelial cells to total gland area was examined, there was a disproportionate (mucous and serous) and the adjacent basement membrane. and significant (p = 0.014) increase for FA compared with the Fine cytoplasmic processes were seen to extend between the NAC group (table 3). The NFA group showed changes in epithelial cells. Myoepithelial cells were only seen in the acini mucous gland area and in the components of the gland that and proximal collecting ducts. They were absent from larger were intermediate between the FA and NAC groups. None of ducts that communicated with the bronchial lumen. In the these differences were significant. No significant association NAC group the myoepithelial cells appeared to be discontinuous between smoking status and myoepithelial actin was found (fig 1B) whereas in the FA group they appeared continuous, among the groups. Both smoking and non-smoking patients thicker and showed patchy layering (fig 1D). The changes in with asthma had equivalent increases in myoepithelial cell area. myoepithelial cells from the NFA group were similar to those In the FA group, 31% of the airway lumen was occupied by seen in the FA group but were less pronounced. mucus which was significantly greater (p = 0.004) than the area http://thorax.bmj.com/

Table 1 Demographic characteristics of the study population on which lung tissue was used for morphometric analysis FA (n = 12) NFA (n = 12) NAC (n = 12)

Sex (M/F)* 6/6 6/6 6/6 Age (years){ 36 (21–51) 35 (18–55) 37 (19–58)

Smoking (S/NS)* 6/6 6/6 6/6 on September 25, 2021 by guest. Protected copyright. Pack years{ 2.5–14 1.25–47.5 9–41.25 Age at asthma onset (years){ 11 (1–44) 17 (2–30) N/A Duration of asthma (years){ 25 (2–43) 19 (2–31) N/A Asthma severity score1 0/1/11 1/6/4 (1 unknown) N/A Oral corticosteroid" 2/4/5 (1 unknown) 10/1/0 (1 unknown) N/A Inhaled corticosteroid" 3/5/3 (1 unknown) 7/2/3 N/A Short-acting bronchodilator" 0/1/11 3/5/4 N/A Time from death to autopsy** 26.3 (5.0–67.5) 26.6 (3.0–58.0) 30.7 (7.0–115.5) Cause of death Asthma Heart disease: 5 Drug overdose: 1 Asphyxia: 1 Heart disease: 3 Substance abuse: 3 Brain tumour: 2 Morbid obesity: 2 Cerebral aneurysm: 1 Diabetes: 1 Musculoskeletal: 1 Seizure disorder: 1 Suicide: 1 Meningitis: 1 Malignant melanoma: 1 *Data expressed as number of patients. {Data expressed as median (range). {Data expressed as range. 1Number of patients: mild/moderate/severe. "Number of patients: none/occasional/everyday use. **Data expressed in hours as mean (range). FA, fatal asthma; N/A, not applicable; NFA, non-fatal asthma; NAC, non-asthma control ; NS, non-smoker; S, smoker.

34 Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from Table 2 Demographic characteristics of subjects from Sao Paulo on ducts. Our results suggest that myoepithelial cells contribute to which lung tissue was used for ultrastructural analysis the abundant mucus that is characteristic of fatal asthma. FA (n = 4) NAC (n = 3) The cause(s) of the disproportionate increase in myoepithelial cell area in fatal asthma are unknown. However, two plausible Sex (M/F)* 3/1 0/3 explanations may be postulated. First, it could be due to an Age (years){ 48 (20–51) 50 (46–51) increased workload required to expel the highly viscous mucus Smoking (S/NS)* 2/2 0/3 characteristic of fatal asthma6820 into the airway lumen. Age of asthma onset (years){ 4 (1–10), unknown N/A Duration of asthma (years){ 40 (10–47), N/A Increased intraduct pressures might also contribute to the 21 1 unknown mucous gland duct ectasia described in this and other studies. Time from death to autopsy{ 12 (6–15) 9.3 (5–14) Second, it may reflect an increased stimulus for mucus secretion Oral corticosteroid1 0/4/0 N/A in asthma in response to asthma triggers. In this regard, it Inhaled corticosteroid1 0/2/2 N/A would be one more component of the airway wall affected by Short-acting bronchodilator1 0/1/3 N/A remodelling. The area of the airway smooth muscle layer is also Cause of death Asthma Heart disease increased in asthma422so that a generalised effect of inflamma- *Data expressed as number of patients. tion, release of growth factors or mechanical influences could {Data expressed as median (range). affect the amount of contractile tissue within the airway wall, {Data expressed in hours as mean (range). 1Number of patients: none/occasional/everyday use. including myoepithelial cells. FA, fatal asthma; N/A, not applicable; NAC, non-asthma controls; NS, non-smoker; S, We assessed the area fraction of myoepithelial cells based on smoker. staining of smooth muscle actin. This gives an estimate of the overall area of myoepithelial cells and, since the thickness of the section is ,10% of the thickness of the myoepithelial cell, the of airway occupied by mucous plugs in the NAC group (6.9%) area fraction provides a good estimate of the volume fraction of but not the NFA group (15.3%, p = 0.068; table 3). The grade of these cells. The increased area and volume fraction of actin mucous gland duct ectasia was significantly greater (p = 0.012) staining suggests an increase in contractile tissue. The two- in the FA group compared with the NAC group. There was no dimensional approach used in this study did not allow us significant effect of smoking status on mucous gland size or accurately to estimate the number or size of individual mucous plug area among the groups. myoepithelial cells and thus we were not able to determine if the changes to the myoepithelial cells resulted from cell DISCUSSION hyperplasia and/or hypertrophy. This awaits a separate study. In this study we show that the increased size of bronchial There are few studies of myoepithelial cells in bronchial mucous glands in fatal asthma is accompanied by a dispropor- mucous glands in humans in the literature.13 To our knowledge, tionate increase in smooth muscle actin in the myoepithelial this is the first study to examine myoepithelial cells in bronchial cells of the glandular acini. These changes were associated with mucous glands of patients with asthma. Myoepithelial cells are http://thorax.bmj.com/ mucous plugs within the airway lumen and ectasia of the gland contractile in nature, possessing myofilaments composed of

Figure 1 (A) Low power (6100) photomicrograph of a bronchial mucous gland from an 18-year-old non-smoking, non-asthmatic female. The section is stained for smooth muscle actin and shows staining of myoepithelial cells on the outer border of the mucous gland on September 25, 2021 by guest. Protected copyright. acini. More intense smooth muscle actin staining is seen of small blood vessels (BV) within and adjacent to the gland. (B) Higher power (6400) view of bronchial mucous gland shown in (A). The myoepithelial cell network (arrows) in the normal appears discontinuous. (C) Low power (6100) photomicrograph of a mucous gland in the bronchial wall of a 42-year-old non-smoking man who died of asthma. The gland is larger than that seen in the non-asthma control (A). The section is stained for smooth muscle actin. There is marked staining of myoepithelial cells within the mucous gland. (D) Higher power (6400) view of centre of gland shown in (C). There is increased staining of myoepithelial cells around the acini. The myoepithelial cells are thicker than in the control (B) and are continuous around the circumference of the acini and in some areas appear multilayered (arrows).

Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 35 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from Figure 2 Ultrastructural aspect of a submucosal gland in a non-smoking non- asthmatic control female patient aged 50 years. (A, B) Myoepithelial cells (mep) lie under the glandular cells (gc) inside the basal lamina (bl). (C) The cytoplasm of the myoepithelial cell has a filamentous appearance (due to the presence of actin and myosin) with dense bodies (arrowheads). The myoepithelial cell is attached to an acinar glandular cell via desmosomes (arrow).

29 actin and myosin, dense bodies, caveolae and elongated cell mediators. Muscarinic receptors, especially the M3 http://thorax.bmj.com/ mitochondria.13 14 23 The mechanism of stimulation varies by receptor subtype, have been implicated in smooth muscle gland type and species. For example, myoepithelial cells from proliferation30 and contractile protein expression in mesenchy- human sweat glands respond to a-adrenergic stimula- mal cells in asthma.31 Whether similar pathogenetic pathways tion24 whereas porcine bronchial submucosal glands contract in occur in the myoepithelial cells of the bronchial submucosal response to acetylcholine.25 In dogs, secretion from mucous-type glands of patients with asthma is not known, but further epithelial cells occurs in response to both b-adrenergic and research on these topics is warranted. cholinergic stimulation.15 In humans, tachykinin mechanisms An increase in bronchial submucosal gland area has been may also be important.26 27 Thus, myoepithelial cell contraction reported as a pathological marker of asthma.5 Our study on September 25, 2021 by guest. Protected copyright. may be caused by cholinergic, b-adrenergic or peptidergic supports this observation. We found a significant increase in mechanisms. mucous gland area as a proportion of airway size in fatal asthma Our findings may be relevant to studies of smooth muscle cell compared with non-asthma controls. Deposition of mucus into function in asthma. Mast cells and neutrophils are increased in the airway lumen is also a prominent feature of fatal asthma, the submucosal glands of patients with asthma.28 The differ- where it is thought to contribute to increased airway entiation and function of myofibroblasts is regulated by mast resistance32 and is readily apparent at autopsy in cases of fatal

Figure 3 Ultrastructural aspect of a submucosal gland in a 45-year-old male smoker with fatal asthma. (A) Myoepithelial cell (mep) with large cytoplasmic branches around the acinar glandular cells (gc). The glandular basal lamina seems to be thickened (bl). (B) Detail of a myoepithelial cell with prominent cytoplasm, rich in myofilaments and showing dense bodies (arrowheads).

36 Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from Table 3 Morphometry of selected airway features in fatal asthma (FA) and non-fatal asthma (NFA) cases and non-asthma control (NAC) subjects Morphometric parameter NAC (n = 12) NFA (n = 12) FA (n = 12) p Value

Airway perimeter, Pbm (mm) 16.6 (1.2)* 15.6 (1.1) 17.1 (1.7) 0.954{ Total mucous gland area{ 128.6 (17.3) 195.4 (28.3) 226.6 (44.4) 0.003{ Mucous plug area/total luminal area1 6.9 (3.2) 15.3 (2.9) 31.0 (7.5) 0.004{ Myoepithelial cell area/mucous gland 10.9 (0.7) 12.2 (0.9) 14.5 (0.9) 0.014{ area1 Grade of mucous gland duct ectasia" 4 (33%) 6 (50%) 11 (92%) 0.012** *Values expressed as mean (SEM). {p Value for FA group compared with NAC group using ANOVA. {These areas were normalised to the basement membrane perimeter (A/Pbm). 1Expressed as a percentage. "Data expressed as number of patients with grade 2 or greater (percentage of patients). **p Value for FA group compared with NAC group using Pearson x2 test.

Table 4 Morphometry of selected airway mucous gland subcomponents in fatal asthma (FA) and non-fatal asthma (NFA) cases and non-asthma control (NAC) subjects Mucous gland subcomponents{ NAC (n = 12) NFA (n = 12) FA (n = 12) p Value

Actin: myoepithelial 14.2 (2.3)* 22.1 (2.7) 32.7 (6.9) 0.017{ Actin: non-myoepithelial1 2.8 (0.5) 3.3 (0.4) 3.3 (0.4) 0.736{ Acinar cell cytoplasm 41.0 (5.4) 63.8 (11.1) 71.9 (15.4) 0.151{ Acinar cell nuclei 4.5 (0.6) 8.7 (1.5) 9.0 (2.3) 0.147{ Acinar lumen 0.6 (0.1) 0.9 (0.3) 2.0 (0.8) 0.118{ Interstitium" 66.1 (9.1) 97.5 (14.2) 109.8 (20.2) 0.120{ *Values expressed as mean (SEM). {Expressed as area/basement membrane perimeter (A/Pbm). {p Value for FA group compared with NAC group using ANOVA. 1Includes arteries, arterioles, veins and lymphatics. "Includes connective tissue, nerves and inflammatory cells. asthma.67 Our study found that luminal mucus content was Funding: Supported by Health and Welfare Canada, Herron Foundation of Alberta, increased in both NFA and FA groups compared with the NAC Alberta Lung Association and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil. AJ is supported by the National Health and Medical group. The FA cases had, on average, 30% of their airway lumen Research Council of Australia. http://thorax.bmj.com/ occupied by mucus compared with approximately 7% in the Competing interests: None. NAC group. Similar findings have been reported by other investigators.728 Ethics approval: Ethics approval was obtained from the institutional review boards at the Universities of Calgary, Alberta, Saskatchewan and Manitoba, Canada. Smoking is also associated with an increase in the size of mucous glands in the conducting airways.33 Smoking evokes a Provenance and peer review: Not commissioned; externally peer reviewed. reflex increase in tracheal submucosal gland secretion in dogs.34 The effect of smoking on myoepithelial cells is largely REFERENCES unknown, but myoepithelial cell ultrastructure was reported 1. World Health Organization. Chronic respiratory diseases. 2008. www.who.int/ on September 25, 2021 by guest. Protected copyright. to be unaffected by smoke exposure in rat tracheal submucosal respiratory/asthma/en/. 35 2. de Magalhaes SS, dos Santos MA, da Silva OM, et al. Inflammatory cell mapping of glands. In the present study, cigarette smoking was not the respiratory tract in fatal asthma. Clin Exp Allergy 2005;35:602–11. associated with an increase in myoepithelial cell smooth muscle 3. James AL, Elliot JG, Abramson MJ, et al. Time to death, airway wall inflammation actin. Our failure to detect an effect of cigarette smoking on and remodelling in fatal asthma. Eur Respir J 2005;26:429–34. 4. James AL, Bai TR, Mauad T, et al. Airway smooth muscle thickness in asthma is myoepithelial cell actin may be related to small sample sizes, the related to severity but not duration of asthma. Eur Respir J 2009;34:1040–5. relatively young age of the population and/or the low to 5. Takizawa T, Thurlbeck WM. Muscle and mucous gland size in the major bronchi of moderate pack years of the cigarette smokers. patients with chronic bronchitis, asthma, and asthmatic bronchitis. Am Rev Respir Dis In summary, we show that excess luminal mucus in fatal 1971;104:331–6. 6. Rubin BK, Tomkiewicz R, Fahy JV, et al. Histopathology of fatal asthma: drowning in asthma is associated with enlarged submucosal glands, increased mucus. Pediatr Pulmonol 2001;(Suppl 23):88–9. myoepithelial cell area and a disproportionate increase in 7. Kuyper LM, Pare PD, Hogg JC, et al. Characterization of airway plugging in fatal myoepithelial smooth muscle actin. These findings add to our asthma. Am J Med 2003;115:6–11. 8. Rubin BK. Physiology of airway mucus clearance. Respir Care 2002;47:761–8. understanding of the complex mechanisms leading to death during 9. Groneberg DA, Eynott PR, Lim S, et al. Expression of respiratory mucins in fatal an acute asthma attack. The results of this study indicate that new status asthmaticus and mild asthma. Histopathology 2002;40:367–73. therapeutic strategies for asthma treatment might include 10. Deugnier MA, Teuliere J, Faraldo MM, et al. The importance of being a medications designed to reduce myoepithelial cell contractility. myoepithelial cell. Breast Cancer Res 2002;4:224–30. 11. Hasegawa M, Hagiwara S, Sato T, et al. CD109, a new marker for myoepithelial cells of mammary, salivary, and lacrimal glands and prostate basal cells. Pathol Int Acknowledgements: The authors greatly appreciate the time and commitment of 2007;57:245–50. the families of the deceased who consented to participate in the study as well as the 12. Li HH, Zhou G, Fu XB, et al. Antigen expression of human eccrine sweat glands. coroners, medical examiners and healthcare workers from the provinces of Alberta, J Cutan Pathol 2009;36:318–24. Saskatchewan and Manitoba. Without their involvement this study would not have 13. Meyrick B, Reid L. Ultrastructure of cells in the human bronchial submucosal glands. been possible. We appreciate the work of Dr Karen Osiowy and Dr Abdel Aziz Shaheen J Anat 1970;107:281–99. for reviewing the design and statistical analyses for this paper. We also thank Artee 14. Shimura S, Sasaki T, Sasaki H, et al. Contractility of isolated single submucosal Karkhanis and Monica Ruff for technical and logistical help with conducting the study. gland from trachea. J Appl Physiol 1986;60:1237–47.

Thorax 2010;65:32–38. doi:10.1136/thx.2008.111435 37 Asthma Thorax: first published as 10.1136/thx.2008.111435 on 8 December 2009. Downloaded from 15. Lung MA. Autonomic nervous control of myoepithelial cells and secretion in 26. Springer J, Groneberg DA, Pregla R, et al. Inflammatory cells as source of of anaesthetized dogs. J Physiol 2003;546:837–50. tachykinin-induced mucus secretion in chronic bronchitis. Regul Pept 16. Hessel PA, Mitchell I, Tough S, et al. Risk factors for death from asthma. Prairie 2005;124:195–201. Provinces Asthma Study Group. Ann Allergy Asthma Immunol 1999;83:362–8. 27. Ballard ST, Spadafora D. Fluid secretion by submucosal glands of the 17. Global Initiative for Asthma (GINA). Global strategy for asthma management and tracheobronchial airways. Respir Physiol Neurobiol 2007;159:271–7. prevention. GINA, 2007. 28. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in 18. James AL, Green FH, Abramson MJ, et al. Airway basement membrane perimeter submucosal mucous glands and mucus plugging in patients with asthma. Thorax distensibility and airway smooth muscle area in asthma. J Appl Physiol 2002;57:677–82. 2008;104:1703–8. 29. Gailit J, Marchese MJ, Kew RR, et al. The differentiation and function of 19. Mauad T, Ferreira DS, Costa MB, et al. Characterization of autopsy-proven fatal myofibroblasts is regulated by mast cell mediators. J Invest Dermatol asthma patients in Sao Paulo, Brazil. Rev Panam Salud Publica 2008;23:418–23. 2001;117:1113–9. 20. Sheehan JK, Richardson PS, Fung DC, et al. Analysis of respiratory mucus 30. Gosens R, Dueck G, Rector E, et al. Cooperative regulation of GSK-3 by muscarinic glycoproteins in asthma: a detailed study from a patient who died in status and PDGF receptors is associated with airway myocyte proliferation. Am J Physiol asthmaticus. Am J Respir Cell Mol Biol 1995;13:748–56. Lung Cell Mol Physiol 2007;293:L1348–58. 21. Cluroe A, Holloway L, Thomson K, et al. Bronchial gland duct ectasia in fatal 31. Gosens R, Zaagsma J, Meurs H, et al. Muscarinic receptor signaling in the bronchial asthma: association with interstitial emphysema. J Clin Pathol pathophysiology of asthma and COPD. Respir Res 2006;7:73. 1989;42:1026–31. 32. James A, Carroll N. Theoretic effects of mucus gland discharge on airway resistance 22. Benayoun L, Druilhe A, Dombret MC, et al. Airway structural alterations selectively in asthma. Chest 1995;107:110S. associated with severe asthma. Am J Respir Crit Care Med 2003;167:1360–8. 33. Chalmers GW, MacLeod KJ, Thomson L, et al. Smoking and airway inflammation in 23. Sanchez-Mora N, Rendon-Henao J, Monroy V, et al. Antigenic profile of human patients with mild asthma. Chest 2001;120:1917–22. bronchial gland. Histol Histopathol 2005;20:865–70. 34. Schultz HD, Davis B, Coleridge HM, et al. Cigarette smoke in lungs evokes reflex 24. Sato K. Pharmacological responsiveness of the myoepithelium of the isolated human increase in tracheal submucosal gland secretion in dogs. J Appl Physiol axillary apocrine sweat gland. Br J Dermatol 1980;103:235–43. 1991;71:900–9. 25. Inglis SK, Corboz MR, Taylor AE, et al. In situ visualization of bronchial submucosal 35. Lewis DJ, Jakins PR. Effect of tobacco smoke exposure on rat tracheal submucosal glands and their secretory response to acetylcholine. Am J Physiol 1997;272:L203–10. glands: an ultrastructural study. Thorax 1981;36:622–4.

Lung alert

Clinical characteristics of the H1N1 virus Prior to 2005, human infections with classic swine influenza viruses were reported at a rate of only one or two cases per year. However, by the late 1990s multiple strains and subtypes (H1N1, H3N2 and H1N2) of triple-reassortant swine influenza A (H1) viruses containing genes from avian, human and swine influenza viruses had emerged and became enzootic among pig herds in http://thorax.bmj.com/ North America. In the first study, 11 sporadic cases of human infections with triple-reassortant swine influenza A (H1) occurring between December 2005 through February 2009 are described using information collected by the Centre for Disease Control in the USA. All but one of the cases definitely had exposure to pigs. Four patients had underlying medical conditions and the median age of the 11 infected was only 10 years. Symptoms included fever, cough, headache, sore throat, diarrhoea and myalgia. All patients recovered. on September 25, 2021 by guest. Protected copyright. In the second study, between 15 April and 5 May 2009, a further 642 confirmed cases of H1N1 are described from 41 states in the USA. In this paper the age range is much broader (3 moths to 81 years). However, the symptom profile is very similar and the spectrum of disease severity ranges from mild self-limiting illness to hospitalisation and death. Both the studies provide a description of symptoms and outcomes of this evolving virus and emphasise the importance of communication and collaboration worldwide to better understand the disease. It is possible that H1N1 infection in humans presents the greatest pandemic threat since the emergence of influenza A (H3N2) virus in 1968. The continued identification of new cases worldwide indicates sustained human to human transmission with great epidemic and pandemic threat to global public health.

c Shinde V, Bridges CB, Uyeki TM, et al. Triple-reassortant swine influenza A (H1) in humans in the United States, 2005–2009. N Engl J Med 2009;360:2616–25. c Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009;360:2605–15.

S A Abbas Correspondence to: Dr S A Abbas, Specialist Registrar, Llandough University Hospital, Vale of Glamorgan, UK; ali.abbas@ doctors.org.uk Thorax 2010;65:38. doi:10.1136/thx.2009.127399

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